ALBERT

Description: Using proteins in a therapeutic context often requires engineering to modify functionality and enhance efficacy. We have previously reported that the therapeutic antileukemic protein macromolecule Escherichia coli L-asparaginase is degraded by leukemic lysosomal cysteine proteases. In the present study, we successfully engineered L-asparaginaseto resist proteolytic cleavage and at the same time improve activity. We employed a novel combination of mutant sampling using a genetic algorithm in tandem with flexibility studies using molecular dynamics to investigate the impact of lid-loop and mutations on drug activity. Applying these methods, we successfully predicted the more active L-asparaginase mutants N24T and N24A. For the latter, a unique hydrogen bond network contributes to higher activity. Furthermore, interface mutations controlling secondary glutaminase activity demonstrated the importance of this enzymatic activity for drug cytotoxicity. All selected mutants were expressed, purified, and tested for activity and for their ability to form the active tetrameric form. By introducing the N24A and N24A R195S mutations to the drug L-asparaginase, we are a step closer to individualized drug design.

Description: Abstract 873 Background. Philadelphia chromosome positive (Ph+) ALL accounts for 3–5% of pediatric ALL. An international survey on 640 children diagnosed between 1995 and 2005 and treated with chemotherapy and stem cell transplantation (SCT) without the use of tyrosin kinase inhibitors (TKI) recently reported an overall 7-year event free survival (EFS) and overall survival (OS) of 31.2% and 44.2%, respectively. In those years, only limited experience was accumulated on the use of Imatinib (IM) for children with Ph+ leukemia. The EsPhALL study was designed as an intergroup, open-label, randomized Phase II/III study, within the I-BFM-SG network, to assess the safety and efficacy of IM in association with chemotherapy. Ten national study groups participated in the study: AIEOP, BFM-G/CH, COALL, FRALLE, NOPHO, MRC, DCOG, CPH, PINDA and HONG KONG. Methods. Patients 1 to 18 years of age diagnosed with Ph+ ALL were eligible to the study. After the induction phase according to national treatment protocol, patients were classified as Good Risk (GR) or Poor Risk (PR) according to their response to treatment. GR patients were those who achieved both the early response (i.e. blast cell count

Description: B-cell precursor childhood acute lymphoblastic leukemia with ETV6-RUNX1 (TEL-AML1) fusion has an overall good prognosis, but relapses occur, usually after cessation of treatment and occasionally many years later. We have investigated the clonal origins of relapse by comparing the profiles of genomewide copy number alterations at presentation in 21 patients with those in matched relapse (12-119 months). We identified, in total, 159 copy number alterations at presentation and 231 at relapse (excluding Ig/TCR). Deletions of CDKN2A/B or CCNC (6q16.2-3) or both increased from 38% at presentation to 76% in relapse, suggesting that cell-cycle deregulation contributed to emergence of relapse. A novel observation was recurrent gain of chromosome 16 (2 patients at presentation, 4 at relapse) and deletion of plasmocytoma variant translocation 1 in 3 patients. The data indicate that, irrespective of time to relapse, the relapse clone was derived from either a major or minor clone at presentation. Backtracking analysis by FISH identified a minor subclone at diagnosis whose genotype matched that observed in relapse ∼ 10 years later. These data indicate subclonal diversity at diagnosis, providing a variable basis for intraclonal origins of relapse and extended periods (years) of dormancy, possibly by quiescence, for stem cells in ETV6-RUNX1+ acute lymphoblastic leukemia.

Description: We have recently defined a new recurrent chromosomal abnormality in patients with acute lymphoblastic leukemia (ALL) involving the duplication of chromosomal band 21q22 and amplification of the AML1 (RUNX1) gene. Currently, fluorescence in situ hybridization (FISH) with probes directed to the AML1 gene is the only reliable detection method. AML1 signals are seen as clusters in interphase cells and in tandem duplication on the long arm of chromosome 21 in metaphase. To date, we have identified 46 patients with this abnormality: 26 males and 20 females. The median age of the 44 children was 9 years (range 4–16 years): two adults were 20 and 30 years old. Their median white cell count was 3.7x109/L (range 1−80x109/L) and all had a common/pre-B immunophenotype. The 3 year event free survival for the 24 children treated on MRC ALL97 was 51% (95% confidence interval 27%–71%). Hence, this abnormality has been associated with older children with a low white cell count and, most significantly, a poor prognosis. The NCRI UK Childhood Leukaemia Working Party has categorized these children into a high-risk group and recommended that they be treated on a more intensive protocol, making their accurate detection vital. To achieve this goal, we have attempted to define the amplified region on chromosome 21 and to identify the genes involved, employing a range of molecular techniques. Microarray analysis (genomic and expression) in conjunction with FISH and quantitative PCR were used to investigate DNA and/or RNA samples from 11 patients. Comparative genomic hybridization (Spectral Genomics 1Mb) (aCGH) showed variable regions of amplification (common minimal region ~10Mb DNA). Complementary FISH, using a range of specific probes corresponding to the aCGH clones, confirmed the extent of the amplified region on chromosome 21, while indicating the precise copy number changes for each clone. FISH also revealed unexpected chromosomal rearrangements of the amplified chromosome, including loss of the 21q telomeric region. These observations confirm the chromosomal instability associated with this abnormality, as previously indicated from the variable chromosome morphology seen by cytogenetic analysis. Expression profiling (Affymetrix HG-U133) detected 60 over-expressed genes in these patients, of which six were located within the chromosome 21 amplicon. This study has defined the extent of the chromosome 21 amplified region surrounding AML1, and highlighted the over-expression of specific genes within the amplicon. This combined molecular approach will help to identify the most appropriate test for the detection of this high-risk abnormality.

Description: Abstract 3303 Introduction: Despite improvement in frontline therapy in childhood acute lymphoblastic leukemia (ALL), central nervous system (CNS) relapse remains a significant clinical problem. The ALLR3 trial (ISCRTN 45724312) was designed specifically to address this issue with the use of drugs known to penetrate the CNS. The trial incorporated a randomization between Mitoxantrone and Idarubicin during induction. Mitoxantrone showed an early benefit in all patients resulting in closure of the randomization in December 2007 (ASH Annual Meeting Abstracts, Nov 2009; 114:3390). Subsequently all patients now receive Mitoxantrone. Here we report on the outcome of patients with isolated CNS relapse (iCNSr) or combined CNS relapse (involvement of CNS and bone marrow, cCNSr). Methods: CNS involvement was defined as ≥5 WBC/μl with morphological evidence of blasts in the cerebrospinal fluid (CSF). Combined relapse (cCNSr) was defined as CNS disease with ≥ 5% blasts in the bone marrow. Time to relapse was classified as, Very Early: within 18 months of first diagnosis; Early: after 18 months of first diagnosis but within 6 months of stopping therapy and Late: more than 6 months after stopping therapy. All patients received 3 blocks of chemotherapy. Subsequently, allogenic stem cell transplant (allo-SCT) was offered to all very early relapses (iCNSr & cCNSr), early iCNSr (irrespective of immunophenotype), all T-cell cCNSr (irrespective of time to relapse) and early or late pre-B cCNSr that had a minimal residual disease level of ≥ 104 at the end of induction. All other patients were offered chemotherapy and cranial radiotherapy. Results: Of a total of 330 relapsed patients, 102 (31%) had CNS involvement. Of these 63 (62%) had iCNSr and 39 (38%) had cCNSr. The incidence of CNS disease was higher in males (M:F, CNS relapses 2.5:1 vs all relapses 1.5:1). CNS relapses had a higher proportion of T-cell disease (pre B:T CNS relapses 3.6:1 vs all relapses 7.8:1]. The number of patients presenting in very early, early and late phases were 19 (19%), 55 (54%) and 28 (27%) respectively. All late iCNSr patients were males. Almost all late relapses (iCNSr and cCNSr) (27/28) were of a pre B phenotype. At the end of induction phase, 91/102 (89%) achieved complete remission (CR) and 82/102 (80%) remained in CR after 3 blocks of chemotherapy. The estimated 3-year overall survival (OS) and progression free survival (PFS) for all patients with CNS disease was 45.5% (95%CI 32.9, 58.0) & 43.4% (95%CI 32.0, 54.7) respectively. There were no significant differences in survival with respect to site of the disease (combined vs isolated), gender or immunophenotype (pre B vs T). As shown in Table 1, CNS relapse patients who received Mitoxantrone had a significantly improved outcome when compared to those who received Idarubicin. This was most evident in those who had i) iCNSr, ii) pre-B phenotype and iii) allo-SCT, when analyzed on an intention to treat basis. This represents a considerable improvement in outcome compared to the results obtained in these sub-groups of patients in the previous UK ALLR2 study (Roy A et.al. Br. J. Haem. 2005;130:67-75). Conclusion: Mitoxantrone is highly effective in children with relapsed pre B ALL who have CNS involvement. As there were no other differences between patients treated on Mitoxantrone or Idarubicin, effective systemic therapy is as important as CNS directed therapy, if not more, in treating patients with CNS relapse. Disclosures: Off Label Use: Most drugs used in this protocol are off label as the majority of drugs used in childhood ALL are not liscensed for use in children.

Description: In ALL, leukemic cells have been shown to modulate the bone marrow microenvironment through aberrant cytokine production favoring leukemic cell survival. Extracellular vesicles have also been recognised as key mediators of non-paracrine cellular interactions in cancer. This mechanism of tumor-stromal interaction has been described in chronic lymphocytic leukemia where recipient stromal cells undergo phenotypic changes. We now report a similar phenomenon in ALL. Light microscopy showed that primary ALL blasts and cell lines, released anucleate extracellular vesicles into extracellular fluids. On transmission electron microscopy, leukemic extracellular vesicles (LEVs) were observed to be heterogeneous, ranging from 100nm exosome-like particles to large 6µm particles. Larger LEVs were enclosed in lipid-rich membranes and contained several organelles including ribosomes, lysosomes, golgi bodies and mitochondria. On fluorescent immunostaining, LEVs demonstrated an organized cytoskeleton with expression of actin, vinculin and talin. On imaging flow cytometry, a relative excess of circulating CD19-positive LEVs were observed in patient samples at diagnosis compared to post-treatment; these were readily distinguished from CD61-expressing platelets. On time-lapse microscopy, LEVs generated by green fluorescent labeled ALL cells, appeared as dynamic particles and were internalized by both leukemic and bone marrow stromal cells. Confocal microscopy revealed internalized labeled LEVs located in the perinuclear region of recipient cells for up to a week. Lipophilic tracer labeled LEVs, ALL cell lines and primary cells were transplanted intrafemorally in NSG mice as independent experiments. Transplanted LEVs were observed in peripheral blood at day 9 of transplantation and in marrow stromal cells in contralateral femurs at day 14 of injection. Bilateral femoral flushes at day 14 in both LEV and ALL xenografts, showed free LEVs in extracellular spaces as well as internalization of LEVs by murine mesenchymal cells. While internalization of LEVs by heterogeneous leukemic cell lines led to phenotypic transformation to the cell of origin, recipient marrow stromal cells did not demonstrate change in phenotype, viability or proliferation. In keeping with this, both control and LEV internalized stromal cells had similar ATP levels. Instead, metabolic analyses using an extracellular flux analyzer indicated that recipient stromal cells demonstrated altered normoxic metabolism, with decreased mitochondrial respiration, and increased extracellular acidification associated with raised lactate production. Thus indicating aerobic glycolysis as the main source of energy. In concordance with this, megakaryocytes, granulocytes and endothelial cells but not lymphoblasts in leukemic murine and human bone marrow demonstrated perimembranous expression of the lactate export protein MCT4 (monocarboxylate transporter 4). In contrast, in normal and remission marrow, while granulocytes express membranous MCT4, endothelial cells do not express MCT4 and megakaryocytes showed a predominant cytoplasmic expression. Thus internalized LEVs triggered a metabolic switch from oxidative phosphorylation to aerobic glycolysis in recipient stromal cells resulting in extracellular lactate generation. We speculate that extracellular lactate in the microenvironment serves as the preferred energy substrate for ALL cells, a phenomenon reported in other cancers and termed the reverse Warburg effect. Targeting lactate-dependent metabolism may therefore represent a novel common therapeutic strategy in ALL and other cancers. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3390 Poster Board III-278 Introduction: While the outcome of children with acute lymphoblastic leukemia (ALL) has steadily improved, the prognosis for those who relapse (rALL) remains poor. Partly this has been due to the lack of a standardised approach to children with rALL. The international collaborative trial ALLR3 stratified patients into standard (SR), intermediate (IR) and high risk (HR) groups based on the time from first diagnosis to relapse, site of relapse and immunophenotype. All patients received 3 blocks of therapy, with a minimal residual disease (MRD) assessment at the end of blocks 1 and 3. Allogeneic stem cell transplantation (allo-SCT) was offered to all HR and those IR who had a MRD of ≥10-4 at week 5. In children in whom MRD was unavailable, allo-SCT was offered to those who relapsed within 24 months of stopping therapy. All other patients continued on chemotherapy for 24 months. A day 1 and 2 randomisation was performed between mitoxantrone and idarubicin. Results: 239 patients were enlisted, of whom 216 were randomised and 109 idarubicin and 103 mitoxantrone patients are analysable. There were non-significant differences between the groups with regards to time to relapse, site of relapse and cytogenetic subtypes.108 (51%) were in CR2 at a median follow up of 36 months (range 1-70) and Progression Free Survival (PFS) at 3 years was 50.3% (95% CI 42.9%, 57.3%). Forty four percent of idarubicin and 40% of mitoxantrone patients had disease levels of ≥10-4 at week 5 (p=0.90) at the end of block 1. The PFS at 3 years for those who received idarubicin was 35.9% (25.9%, 45.9%) and mitoxantrone 64.6% (54.2%, 73.2%) (p=0.0004). Adjusted for differences in risk group, country, age, gender and cytogenetic subtype, this difference continues to be significant (p=0.003). Overall mitoxantrone was better tolerated and less toxic than idarubicin. A competing risks model showed that the difference between the two drugs is primarily related to disease control (p=0.02), rather than toxicity of treatment (p=0.09). In the patients who were transplanted, 35% and 5% of patients who received idarubicin (n = 48) or mitoxantrone (n = 44) respectively have relapsed. Based on the intention to treat analyses, there were non significant differences in patients treated without an allo-SCT in both arms. IR patients who were transplanted did worse (p = 0.01) in the idarubicin group but had comparable results in the mitoxantrone group to those who received chemotherapy. As a result the randomisation in ALLR3 has now been closed, though the study continues to accrue to better answer the MRD stratification question. Conclusions: Mitoxantrone is a well tolerated and highly effective drug for the treatment of children with rALL. While MRD at week 5 failed to predict the differences in outcome for the two randomised groups, it identified that children in the IR group with

Description: Abstract 3245 Whilst modern treatment approaches cure a high number of patients with acute lymphoblastic leukemia (ALL), little progress has been made in the treatment of refractory and relapsed ALL and new treatment approaches are needed. We recently demonstrated that anti-HLA-DR class II monoclonal antibody (mAb) L243 induces a novel non-apoptotic mode of cell death in B-cell lymphoma lines, defined by homotypic adhesion (HA), actin reorganisation and lysosomal activity (Ivanov et al. J Clin Invest, 2009). Here, we extend these important observations and examine whether this novel form of mAb induced cell death occurs in pre-B ALL cell lines. Expression of HLA-DR was determined using flow cytometry in a panel of ALL cell lines (REH, SupB15 and SD1). HLA-DR was expressed at high levels on each of the cell lines. The ability of L243 to induce HA and cell death (Annexin V/PI positivity) was assessed using microscopy and flow cytometry respectively. L243 was able to evoke both strong HA and cell death in all of the ALL cell lines (e.g. in SupB15 cells 46±1.7% death versus 7±0.5% in controls, p